1. No category

A Genetic Screen for Genes Regulating the Generation of
Drosophila Intermediate Neural Progenitor Cells
Thien Luu, Yonggang Xie, Xiaosu Li and Sijun Zhu
DEPARMENT OF NEUROSCIENCE AND PHYSIOLOGY, SUNY UPSTATE MEDICAL UNIVERSITY, SYRACUSE, NEW YORK
ABSTRACT
MATERIALS & METHODS
 Fly genetic crosses to make haploinsufficient flies
for screening
Conclusion
1. 10 Df lines on the second and third chromosomes were identified to show the suppression of Btd
RNAi knockout phenotype. This suppression led to the rescue of mature INPs in all of type II NB
lineages in Drosophila larval brains
pntP1-Gal4 > UAS-mCD8-GFP
GFP Dpn
Number of mature INPs
30.00
A
wt
UAS-Btd RNAi
C
C
df 8089/+, UAS-Btd RNAi
25.00
*
*
20.00
*
*
*
*
*
**
*
*
Future Directions
10.00
5.00
0.00
Btd RNAi
120
100
*
*
*
*
*
*
*
*
*
*
60
40
20
0
B’
A’
E
C’
Btd RNAi
(A-A’) All wild-type type II NB lineages contain several mature INPs.
(B-B’) Btd RNAi knockout larval brains show a loss of mature INPs in about 70% of type II NB lineages.
(C-C’) Haploinsufficient deficient line 8089 rescues the loss of mature INPs in all type II NB lineages caused by Btd RNAi knockout.
(D) Quantification of the number of mature INPs in larvae with indicated genotypes. * and **, P < 0.01 and 0.05, respectively, compared with Btd RNAi.
(E) Quantification of the percentage of type II NB lineages containing mature INPs in larvae with indicated genotypes. *, P < 0.01 compared with Btd RNAi.
2. Screening revealed 6 Df lines on the second and the third chromosomes that enhanced the Btd RNAi phenotype in
Drosophila larval brains. The enhancement of Btd RNAi phenotype resulted in an increase in the loss of mature INPs
in type II NB lineages.
GFP Dpn
pntP1-Gal4 > UAS-mCD8-GFP
30.00
www.PosterPresentations.com
A’
UAS-Btd RNAi
B
df 9064/+, UAS-Btd RNAi
*
25.00
20.00
15.00
10.00
5.00
*
*
C
*
*
*
*
*
*
Btd RNAi
120
Percentage of Type II NB Lineages
Containing INPs (%)
A
Dpn
RESEARCH POSTER PRESENTATION DESIGN © 2012
*
80
0.00
 Third instar larvae with desired phenotype were
selected for examination using immunostaining and
confocal microscopy
**
B’
D
100
*
80
60
40
20
This screen identified multiple haploinsufficiency
loci that suppress or enhance the INP loss
phenotypes of Btd RNAi knockdown. Findings
from this screen will allow us to identify novel
genes that regulate the generation of INPs in
type II NB lineages in Drosophila larval brains.
15.00
D
Percentage of Type II NB Lineages
Containing INPs (%)
Dpn
B
Number of mature INPs
Intermediate neural progenitor (INP) cells which are
generated from neural stem cells have the ability to renew
themselves and to produce differentiated neurons.
Mammalian INPs play an important role in the production
of large and complex brain structure. The identification of
INPs in Drosophila type II neuroblast (NB) (Drosophila
neural stem cells) has provided an excellent model for INP
research for the past few years. In order to achieve their
functions, INPs must retain their proliferative ability by
preventing differentiation and cell cycle exit as well as
maintain their specific developmental fate. Our lab has
identified that a homolog of mammalian Sp8 transcription
factor Buttonhead (Btd) plays a critical role in averting
premature differentiation and cell cycle exit of INPs in
Drosophila larval type II neuroblast lineages. The loss of
Btd leads to the elimination of mature INPs in type II NB
lineages, but Btd prevents premature differentiation of INP
remains to be elucidated. Here, we screened
haploinsufficient loci that enhance or suppress the loss of
INP phenotypes caused by Btd RNAi knockdown by using
deficient (Df) lines. Knockdown of Btd results in
elimination of INPs in about 70% of type II NB lineages,
thus providing a sensitized genetic background for the
screen. We screened total 376 deficiency lines of the
second and the third chromosomes and found that 10 lines
that suppress and 6 lines that enhance the loss of INP
phenotypes caused by Btd RNAi knockdown. This initial
screen will allow us to identify genes that function in the
Btd pathway to regulate the generation of Drosophila INPs
in the future.
Results
*
*
*
*
0
Btd RNAi
(A-A’) Btd RNAi knockout larval brain shows 70% loss of mature INPs in type II NB lineages.
(B-B’) Deficient line 9064 enhances the Btd RNAi knockout phenotype, leading to a complete loss of mature INPs in all type II NB lineages.
(C) Quantification of the number of mature INPs in larvae with indicated genotypes. *, P < 0.01 compared with Btd RNAi knockout phenotype.
(D) Quantification of percentage of type II NB lineages containing mature INPs in larvae with indicated genotypes. *, P < 0.01 compared with Btd RNAi.
 Further screening the X and the fourth
chromosomes to identify any more genes involved
in the Btd pathway.
 Narrow down the screening in order to identify
genes that function in Btd pathway to regulate
the generation of INPs by using smaller deficient
lines and specific gene mutant.
 After identifying involved genes, examine the
functions and how those genes are involved in the
Btd pathway.
REFERENCES
Xie, Y., Zhan, X., Li, X., Mei, S., Li, H., Urso, A.and Zhu, S.
(submitting manuscript). The Drosophila Sp8 transcription
factor Buttonhead prevents premature differentiation of
intermediate neural progenitors.
Homem, C. C., and Knoblich, J. A. (2012). Drosophila neuroblasts:
a model for stem cell biology. Development 139, 4297 – 4310.
Chia, W., Somers, W.G., and Wang, H. (2008). Drosophila
neuroblast asymmetric divisions: cell cycle regulators,
asymmetric protein localization, and tumorigenesis. J. Cell
Biol, 180, 267–272.
Zhu, S., Wildonger, J. Barshow, S., Younger, S., Huang, Y., and Lee,
T. (2012). The bHLH Repressor Deadpan Regulates the Selfrenewal and Specification of Drosophila Larval Neural Stem
Cells Independently of Notch. PLoS One 7, e46724.
Bello, B.C., Izergina, N., Caussinus, E., and Reichert, H. (2008).
Amplification of neural stem cell proliferation by intermediate
progenitor cells in Drosophila brain development. Neural Dev 3,
5.
Boone, J. Q., and Doe, C. Q. (2008). Identification of Drosophila
type II neuroblast lineages containing transit amplifying
ganglion mother cells. Dev Neurobiol 68, 1185 – 1195.
Zhu, S., Barshow, S., Wildonger, J., Jan, L. Y., and Jan, YN.
(2011). Ets transcription factor Pointed promotes the
generation of intermediate neural progenitors in Drosophila
larval brains. Proc Natl Acad Sci USA 108, 20615 – 20620.
FUNDING
This project is supported by March of Dimes Basil
O’Connor Starter Scholar Research Award (S.Z., #5FY14-59), SUNY Upstate Medical University startup fund
(S.Z.), and SUNY Upstate Medical University SURF
program.